Immunoassay and related techniques have become the norm for determination of various analytes in biological samples. A variety of formats designed to simplify and improve the accuracy of these tests is available in the art, and the number represented by this variety of formats is very high.
The success of these assays rests in the ability to provide specific binding reagents, usually antibodies or fragments of antibodies, which are highly specific for the target analyte with respect to additional possible components of the sample. For example, there are a large number of assays on the market for pregnancy which rely on the detection of human chorionic gonadotropin (HCG) in urine. These assays are capable of HCG detection because the antibodies provided, which are immunoreactive with HCG, do not react to any detectable extent with other urinary components.
In certain other contexts, however, it is desired to analyze samples for analytes which are members of groups that are cross-reactive with antibodies prepared against any one of them, and any or a number of which may be present in the same sample. One example of this problem relates to the efforts to determine pesticides and herbicides in the environment, since many of these materials are structurally similar. See, for example, van Emon, J. N., et al., in "Analytical Methods for Pesticides and Plant Growth Regulators: Advanced Analytical Techniques," Sherma J. ed., Academic Press, New York, 1989, pp. 217-263; Vanderlaan, M., et al., Environ Sci Technol (1988) 22:247-254; Newsome, W. H., J Assoc Offic Anal Chem (1986) 69:919-923.
Typically, it will not be known for certain which of the several members of a particular class of pesticides, for example the carbamate pesticides, will be present in the environment; in addition, degradation products of the pesticide actually applied may also cross-react with a purportedly specifically immunoreacting antibody or other binding agent. Thus, it will not be possible, in a simple single antibody assay to obtain a reliable picture of the composition of the sample. Indeed, the results of such assays are often given in terms of "equivalents" of a particular identified member of the class to which the antibody, for example, has been prepared. In addition to the cross-reactivity of the possible analytes for any specific binding reagent created against one of them, the concentration ranges of these compounds are very low in typical determinations, typically 10-100 nM, or in the parts per billion range. At these low concentrations, problems of crossreactivity with more abundant materials are particularly troublesome.
Because of the cross-reactivity discussed above, it is difficult to make a definitive determination of analyte concentration. For example, suppose an antibody has 100 times the affinity for analyte B as for analyte A. It would not be possible to distinguish, using a single determination with that antibody, a 50 nM concentration of analyte A from a 0.5 nM concentration of analyte B. Various mixtures of A and B would also react in a quantitatively identical manner. Thus there is no mechanism to use a single antibody for assaying samples that contain mixtures of various structurally similar analytes.
The present invention overcomes these difficulties by utilizing mathematical pattern recognition techniques applied to panels of binding agents with overlapping specificities. Once a set of standard binding patterns for target analytes is determined, more reliable determination of analyte composition in experimental samples becomes possible with concomitant improvement in the accuracy of analyte quantitation.